Sirolimus is a carbocyclic lactone-lactam macrolide produced by the bacterium streptomyces-hygroscopicus. In addition to its antibiotic and antifungal properties, sirolimus acts as a potent immunosuppressive agent. Indeed, sirolimus is frequently used to suppress immune function following transplant procedures. Sirolimus also has antiproliferative properties and has been used recently in conjunction with cardiac stents to prevent restenosis at treatment sites within body vessels.
Considering the widespread and growing use of sirolimus as a therapeutic agent in a variety of clinical procedures, the abilities to accurately detect sirolimus in a sample, such as a patient sample, and to determine concentrations of the compound have increasing importance. For example, it may be desirable to monitor concentrations of the compound in blood to ensure that an effective concentration has been reached and/or is being maintained. If necessary, adjustments to treatment dosages can be made based on the concentrations determined in an appropriate assay.
Current assays for the determination of sirolimus in whole blood samples include an assay based on high-performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques. While HPLC and MS techniques may provide desired accuracy and sensitivity, these assays require specialized skill and equipment. Typically, these techniques are not conducted at clinical facilities and samples are often sent to independent laboratories for analysis, which introduces a delay into the testing program.
Immunoassay techniques, which use antibodies for the detection of an analyte in a sample, are relatively simple laboratory procedures and can often be conducted using automated equipment. Indeed, clinical laboratories typically have such equipment on-site, making immunoassays a desirable format for the analysis of patient samples.
Prior art immunoassays for the determination of sirolimus require the use of an antibody generated using the whole sirolimus compound. This may produce undesirable results because antibodies generated in this manner may cross-react with sirolimus metabolites that have little to no immunosuppressive or other therapeutic activity. For example, sirolimus is metabolized by cytochrome p450 3A enzymes to produce an array of metabolites, including demethylated metabolites that have negligible immunosuppressive activity. These metabolites can be present in patient samples at significant levels (as much as 10% for 16-0-demethyl and 39-0-demethyl species), making any cross-reactivity potentially significant.
Accordingly, there is a need for improved methods and kits for the determination of sirolimus in samples.